Mucus glycoproteins are the major constituents of the mucosal layer found in the upper and lower airways of mammalian lungs Rose. M. C. (1992), Am. J. Physiol. 263:L413-LA29!. Mucus confers the viscoelastic properties upon airway secretions and thus provides for the lubricative and protective qualities necessary for optimal mucocilliary clearance of foreign material from the lungs Lamblin, G., et al. (1992) Eur. Resp. J. 5:247-256!. In certain disease states such as cystic fibrosis Rose, M. C. (1988) Horm. metabol. Res. 20:601-609!, acute and chronic bronchitis Reid, L. (1960) Thorax 15:132-141!, and asthma Aikawa, T., S. Shimura, (1992) Chest 101:916-921!, a situation exists where there is a hypersecretion of mucus glycoproteins from the airway secretory cells of afflicted patients. Hypersecretion of mucus glycoprotein into the luminal airway space may result in obstruction of bronchiolar passages and provide a milieu for bacterial colonization Rose, M. C. (1992) Am. J. Physiol. 263:L413-L429!. Thus, an excess presence of mucus may lead to impaired mucocilliary clearance, progressive respiratory insufficiency, and may ultimately contribute to the morbidity and mortality associated with the above mentioned diseases.
The underlying neural and cellular mechanisms which contribute to the metaplastic changes of the airway epithelial cells that lead to mucus hypersecretion are not well understood. In order to study these mechanisms, investigators have subjected small animals to irritants such as endotoxin Stolk, J., A. (1992) J. Pathol. 167:349-356; Harkema, J. R. and J. A. Hotchkiss. (1992) Am. J. Pathol. 141:307-317!, SO.sub.2 Lamb, D. and L. Reid. (1968) J. Path. Bact. 96:97-111; Jany. B., (1991) Biochem. Biophys. Res. Comm. 181:1-8!, ozone Pino, M. V., et al. (1992) Am. Rev. Respir. Dis. 145:882-889!, and cigarette smoke Farley, J. M. (1992) Annu. Rev. Pharmacol. Toxicol. 32:67-88!. Histological studies reveal that endotoxin or SO.sub.2 exposure causes a profound increase in the number of mucus containing cells which line both the upper and lower airways of the lung Stolk, J., et al. (1992) J. Pathol. 167:349-356; Harkema, J. R. and J. A. Hotchkiss (1992) Am. J. Pathol. 141:307-317; Lamb, D. and L. Reid (1968) J. Path. Bact. 96:97-111!. The increase in airway mucus is demonstrated by an increase in number of Alcian blue/Periodic Acid Schiff s (PAS) staining of airway epithelial cells, and by an elevation in .sup.35 SO.sub.4 uptake into explanted airways.
Tracheal lavage samples from endotoxin treated rats also show an increased presence of mucin material as deduced by immunoreactivity with specific monoclonal antibodies Steiger, D. J., et al. (1993) Am. Rev. Resp. Dis. 147:A437 (abstr.)!. Aside from examining increases in the amounts of mucus glycoprotein through the use of histological and immunological techniques, Basbaum and coworkers, (1991), Biochem. Biophys. Res. Comm. 181:1-8! have also shown that substantial increases in mRNA coding for mucin are associated with mucus cell metaplasia. Their data suggests that exposing rats to endotoxin Steiger, D. J., et al. (1993) Am. Rev. Resp. Dis. 147:A437 (abstr.)! or SO.sub.2 Jany, B., (1991). Biochem. Biophys. Res. Comm. 181:1-8! initiates mucin gene transcription resulting in the de novo synthesis of mucus glycoprotein by the airway epithelia cell.
Animal Models of Mucus Hypersecretion
Hypersecretion of mucus glycoproteins by secretory airway epithelial cells in patients afflicted with cystic fibrosis Rose, M. C. (1988) Horm. metabol. Res. 20:601-608!, asthma Aikawa, T., (1992) Chest 101:916-921!, and chronic bronchitis Reid, L. (1960) Thorax 15:132-141! is thought to contribute to the morbidity and mortality associated with those disease states Rose, M. C. (1992) Am. J. Physiol. 263:L413-L429!. Histological sections prepared from airway tissue removed from subjects afflicted with airway disease at time of autopsy exhibit mucus plugging of distal airways, increased numbers of mucus containing goblet cells Aikawa, T., et al. (1992) Chest 101:916-921!, and an enlargement of submucosal glands Douglas, A. N. (1990) Thorax 35:198-201!.
The basis for airway obstruction with mucus is unclear, but the concepts of hypersecretion and reduced mucocilliary clearance are two viable explanations. An increase in the number of cells capable of synthesizing and secreting mucus glycoproteins may certainly contribute to the observed mucus plugging of airway passages. Biochemical studies on mucus glycoprotein from individuals with airway disease have demonstrated alterations in sialylation and sulfation of the oligosaccharide chains attached to the mucin protein core Cheng. P., et al. (1989) J. Clin. invest. 84:68-72; Frates, R. C., et al. (1983) Pediatr. Res. 17:30-34; Mawhinney, T. P., et al. (1992). Carb. Res. 235:179-197!. Changes in the physicochemical nature of mucus in the disease state might impede its removal from the airways by mucocilliary clearance mechanisms, thereby allowing a buildup of material in the airways and eventual plugging.
Numerous laboratories have vigorously pursued the mechanisms underlying the metaplastic changes seen in the secretory airway cells and changes in the physicochemical make-up of secreted mucins. Small animals have been exposed to environmentally relevant irritants that are believed to be causal components in the development of airway disease in man. Models that have been and are still currently used for the study of chronic obstructive pulmonary disease are exposure of animals to SO.sub.2 Lamb, D. and L. Reid. (1968). J. Path. Bact. 96:97-111; Jany. B., et al. (1991). Biochem. Biophys. Res. Comm. 181:1-8!, ozone Pino, M. V., et al. (1992). Am. Rev. Respir. Dis. 145:882-889!, cigarette smoke Farley, J. M. (1992). Annu. Rev. Pharmacol. Toxicol. 32:67-88! and endotoxin Stolk, J., (1992). J. Pathol. 167:349-356; Harkema, J. R. and J. A. Hotchkiss. (1992). Am. J. Pathol. 141:307-317!. Histological studies performed on these models all demonstrate hypertrophic and hyperplastic changes in the mucus secreting cells lining the airways and, as well, in the submucosal layers of the upper airways. Such changes are comparable to those seen in the above mentioned airway diseases. The neurogenic and humoral mediators responsible for hypersecretion have not been identified todate.
Techniques for Investigating Airway Mucus Cell Metaplasia
Investigators have relied heavily on histological methods for examining changes in airway tissue following irritant exposure Harkema. J. R. and J. A. Hotchkiss. (1992). Am. J. Pathol. 141:307-317!. Their studies have given detailed accounts of changes in alveolar airspace, airway lumen caliber, basement membrane thickness, cellular infiltration, epithelial sloughing, and size and numbers of mucus containing cells. Use of PAS and Alcian blue staining has allowed for quantitative examinations into the up-regulation of acidic and neutral mucus glycoproteins at different levels in the airway Lamb, D. and L. Reid. (1968), J. Path. Bact. 96:97-111!.
Histological methods, although yielding exquisite detail of changes occurring in the airways, are extremely tedious, time consuming and require a high level of expertise for documenting consistent and reproducible results.
In conjunction with histological methods, investigators have injected radioactive sulphate into animals to examine levels of sulphomucins in the airway cells following SO.sub.2 exposure Lamb, D. and L. Reid. (1968). J. Path. Bact. 96:97-111!. More recently Basbaum and coworkers have used monoclonal antibodies that specifically recognize rat goblet and submucosal cell secretory products Finkbeiner, W. E. and C. B. Basbaum. (1988). Am. J. Path. 131:290-297!.
Tracheal lavage samples from rats given intratracheal instillations of endotoxin show an elevated release of macromolecules into the airway space compared to control. Such studies have extended into transcriptional aspects of mucin gene expression through the use of specific cDNA probes which recognize mRNA coding for mucus glycoprotein. Elevations in mucin mRNA were observed in trachea from animals treated with endotoxin Steiger, D. J., (1993) Am. Rev. Resp. Dis. 147:A437 (abstr.)!, and sendei virus infected rats exposed to SO.sub.2 Jany, B., (1991). Biochem. Biophys. Res. Comm. 181:1-8!.
The use of specific antibodies and cDNA probes are particularly useful for examining the up-regulated expression and hypersecretion of specific forms of mucus glycoprotein. However, potential drawbacks for the use of specific antibodies and cDNA probes in exploring various models of hypersecretion are that the available probes may be species, and/or model specific. In addition, the availability of such probes may be limited, especially for investigators unable to develop their own antibodies or cDNA. Such limitations may ultimately hinder the advancement of knowledge concerning the mechanisms leading to airway hypersecretion.
Therefore, in order to examine metaplastic changes in airway mucus secretory cells, investigators have relied heavily on the histological methods which are tedious and are at best semiquantitative. As mentioned above, immunological and molecular biological techniques have been employed by some laboratories to study mucus gene expression and hypersecretion. These techniques are based on the availability of specific antibodies or cDNA probes that recognize the type(s) of mucin which is up-regulated for that particular animal species, strain, and irritant model. The instant invention is a method for the rapid estimation of hyperplastic and hypertrophic changes in animal airways, by directly measuring mucins, regardless of the model, strain, or species, as opposed to indirect measurement of mRNA.